1. Technical Field
This invention pertains to the detection and quantification of biomolecules by hybridization assay, and pertains more particularly to hybridization assays wherein reporter molecules are used for signal amplification.
2. Background
Nucleic acid hybridizations are now commonly used in genetic research, biomedical research and clinical diagnostics to detect and quantify particular nucleotide sequences which are present in heterogeneous mixtures of DNA, RNA, and/or other materials. In the basic nucleic acid hybridization assay, single-stranded analyte nucleic acid (either DNA or RNA) is hybridized, directly or indirectly, to a labeled nucleic acid probe, and the duplexes containing label are quantified. Both radioactive and nonradioactive labels have been used.
The basic assay lacks sensitivity. When the analyte is present in low copy number or dilute concentration the signal cannot be distinguished from the background noise. Variations of the basic scheme have been developed to facilitate separation of the target duplexes from extraneous material and/or to amplify the analyte sequences in order to facilitate detection, but these variations have suffered generally from complex and time consuming procedures, high background, low sensitivity, and difficulty in quantification. A primary object of the present invention is to provide an amplifier for use in hybridization assays that provides a highly reproducible gain in signal, a highly reproducible signal-to-noise ratio, is itself quantifiable and reproducible, and is capable of combining specifically with an analyte present at low concentration and with a "universal" reporter moiety to form a stable complex.
Commonly owned U.S. Pat. No. 4,868,105, issued 19 Sep. 1989, the disclosure of which is hereby incorporated by reference, describes a solution phase hybridization sandwich assay in which the analyte nucleic acid is hybridized to a "labeling probe" and to a "capturing probe". The probe-analyte complex is coupled by hybridization to a solid-support. This permits the analyte oligonucleotide to be removed from solution as a solid phase complex, thereby concentrating the analyte, facilitating its separation from other reagents, and enhancing its subsequent detection.
PCT Application 84/03520 and EPA 124221 describe a DNA hybridization assay in which: (1) analyte is annealed to a single-stranded DNA probe that has a tail that is complementary to an enzyme-labeled oligonucleotide, and (2) the resulting tailed duplex is hybridized to an enzyme-labeled oligonucleotide. The Enzo Biochem "Bio-Bridge" labeling system appears to be similar to the system described in these two patent applications. The "Bio-Bridge" system uses terminal deoxynucleotide transferase to add unmodified 3'-poly T-tails to a DNA probe. The poly T-tailed probe is hybridized to the target DNA sequence and then to a biotin-modified poly A.
EPA 204510 describes a DNA hybridization assay in which analyte DNA is contacted with a probe that has a tail, such as a poly dT-tail, and an amplifier strand that has a sequence, e.g., a poly dA sequence, that hybridizes to the tail of the probe and is capable of binding a plurality of labeled strands.
The main problem with these prior hybridization assays is that they lack sufficient specificity and/or signal to be useful for detecting very low levels of analyte.
Another commonly owned EP Application No. 88309697.6 (publication No. 0317077), filed 17 Oct. 1988, the disclosure of which is hereby incorporated by reference, describes linear and branched oligonucleotides which can be used as a signal amplifiers in hybridization assays. Here the amplifier oligomer has two domains--a first domain which is complementary to a target sequence (either the analyte per se or a "linker probe") and a second domain, present in repeating units, complementary to a labeled reporting sequence. The multiplication of reporting sequences per target sequence provides for the amplification of the signal.
Another approach has been to use nucleic acid polymerases to amplify target sequences. For example, the so-called polymerase chain reaction (PCR), uses repeated cycles of DNA primed, DNA-directed DNA polymerase synthesis to amplify sequences of interest (Saiki, R. K., et al., Science (1986) 230:1350-1354). The amplified target is then detected using the basic hybridization assay protocol.
RNA polymerases have also been used to amplify target sequences (Krupp, G., and Soll, D. FEBS Letters (1987) 212:271-275). This approach has been incorporated into a hybridization format that involves production of a double-stranded copy of the target sequence, insertion of a RNA polymerase promoter sequence, transcription of the copy and detection by hybridization assay (Kwoh, D. Y., et al., Proc. Natl. Acad. Sci. U.S.A. (1989) 86:1173-1177). Since DNA-directed RNA polymerases produce up to 103 copies of RNA per copy of DNA template, fewer cycles of amplification are required. Bacteriophage DNA-dependent RNA polymerases (e.g., T3, T7, SP6) have previously been employed for the preparation in vitro of specific RNA sequences from cloned or synthetic oligonucleotide templates and are well understood (Melton, D. A., et al., Nucleic Acids Res. (1984) 12:7035-7056); Chamberlin, M. and Ryan, T., (1982) in "The Enzymes," Boyer, P. D., ed., 15:87-108; Martin, C. T., and Coleman, J. E., Biochemistry (1987) 26:2690-2696). These polymerases are highly promoter specific. DNA sequences from 17 T7 promoters are known and a consensus sequence has been deduced (Oakley, J. L., and Coleman, J. E., Proc. Natl. Acad. Sci. U.S.A. (1977) 74:4266-4270; Dunn, J. J., and Studier, F. W., J. Molec. Biol. (1983) 166:477-535). It is also known that to retain polymerase activity, only the promoter region must be double-stranded (Milligan, J. F., et al., Nucleic Acids Res. (1987) 15:8783-8799).
Finally, RNA-directed RNA polymerase has also been used to detect target sequences (Lizardi, P. M., et al., Bio/technology (1988) 6:1197-1202; Lomeli, H., et al., Clin. Chem. (1989) 35:1826-1831). In this system an RNA probe is prepared by coupling RNA complementary to the target sequence with RNA (MDV-1) (U.S. Pat. No. 4,786,600) which serves as an exclusive template for the bacteriophage Q-beta (Q) replicase. First, the target is immobilized on a solid substrate, then the RNA probe is hybridized to the target and finally the probe is eluted. Subsequent addition of Q-beta-polymerase to the probe generates multiple copies of the template/target RNA. In a related assay, MDV-1 RNA was first bound to biotin, then coupled to an avidinylated target, and subsequently assayed as described above (Chu, B. C. F., et al., Nucleic Acids Res. (1986) 14:5591-5603).
The use of Q-beta-replicase in hybridization assays has four major disadvantages:
1) Q-beta-replicase is typically contaminated with MDV-1 RNA. Consequently, this system has very high background (poor signal-to-noise ratio) when the reporter sequence is the MDV-1 sequence itself; PA1 2) The probe is RNA. RNA is highly sensitive to degradation from the RNAase activity which is ubiquitous in crude cellular preparations, and from the alkaline conditions required to denature double-stranded DNA targets; PA1 3) Due to the secondary structure of MDV-1 RNA there is considerable nonspecific binding in hybridization assays, thus significantly lowering the sensitivity of the assay and precluding accurate quantification; and, PA1 4) The amount of signal (the RNA product of Q-beta-replicase) varies with the log of the number of probes originally bound to the target. Thus, this assay can only detect order-of-magnitude differences between the concentrations of analyte in various samples. PA1 (i) a first domain (A) which is single-stranded and has a nucleotide sequence (a') complementary to a target sequence (a) (FIG. 2A) the target sequence comprising a domain either within the analyte sequence or within the sequence of an oligonucleotide which also contains a sequence domain complementary to the analyte sequence; PA1 (ii) a second domain (B) which is double-stranded and capable of function as a promoter for a DNA-dependent RNA polymerase enzyme activity; and PA1 (iii) a third domain (C) which is either single- or double-stranded and adjacent to the second domain, such that the third domain is capable of functioning as a template (c') for the promoter activity of the second domain (FIG. 2B). PA1 (i) immobilizing the analyte, directly or indirectly, on a solid substrate; and hybridizing the polydeoxynucleotide template probe described supra, directly or indirectly, to the analyte; PA1 (ii) next removing the unhybridized template probe; PA1 (iii) next transcribing (via a DNA-dependent RNA polymerase activity) multiple copies of RNA oligomers (c) which are complementary to the template sequence (c') of domain C of the amplifier; and PA1 (iv) finally quantifying the RNA transcripts.
The invention disclosed herein has several advantages over the Q-beta-replicase method. First, the probe is DNA rather than RNA. Second, the assay has very high signal to noise ratio and very high sensitivity. Third, since the signal is amplified rather than the target, the oligomer which is actually measured will always have the same sequence and size, thereby enabling the standardization and optimization of assay conditions (in addition, most of the biological reagents can be used universally thereby further simplifying and standardizing the assay). Finally, the target can be easily and accurately quantified.